CN104403017B - The preparation method of a kind of low viscosity, high purity yeast mannans - Google Patents

Abstract

The invention provides the preparation method of a kind of low viscosity, high purity yeast mannans, with brewing yeast cell wall for raw material, adopt water extraction and alcohol precipitation method, obtain crude product yeast mannans, centrifugal through neutral protease and PRONASE A enzymolysis, reprocess and get final product.The present invention is by combining secondary enzymolysis, centrifugal, ultrafiltration and spray drying technology, the equipment adopted all can realize the needs of suitability for industrialized production, the yeast mannans purity of gained is greater than 90%, viscosity is low and be Newtonian fuid, can not affect the rheological properties of system in actual applications.

Description

A kind of preparation method of low-viscosity, high-purity yeast mannan

technical field

The invention relates to the preparation of yeast mannan, in particular to a method for preparing yeast mannan with low viscosity and high purity.

Background technique

Yeast mannan is a kind of yeast cell wall polysaccharide, which accounts for about 40% of the dry weight of yeast cell wall. It is composed of main chain and side chain. There are mannose side chains connected by α-1,2 or α-1,3 bonds, which are connected to proteins through O-glycopeptide bonds and N-glycopeptide bonds, consisting of 5% to 20% proteins, 80% to 90% of the mannose composition has a molecular weight of about 20KDa to 200KDa. Mannan has a variety of immunological functions, such as regulating the balance of intestinal flora, enhancing immunity, binding or adsorbing mycotoxins, anti-oxidation, etc., and has broad application prospects.

Yeast mannan is linked to protein in the form of covalent bonds in the yeast cell wall. The mannan obtained by crude extraction contains more protein and has a lower purity. Using appropriate methods to reduce the protein content is the key to the preparation of high-purity yeast mannan The key, at present, the methods for removing protein mainly include: Sevage method, trichloroacetic acid method and calcium chloride method, etc. However, these methods have the following problems: high protein removal rate and low polysaccharide retention rate, organic solvent introduction and residue, water The phase system is not suitable, which is not conducive to the improvement of the purity of yeast mannan.

Yeast mannan in the domestic market is mainly in specifications with a content of 20% to 50%, the price is low, and the low purity limits the wide application of yeast mannan. Therefore, research and development of a method to improve the purity of yeast mannan, It has important economic and social benefits for improving the added value of yeast mannan products and meeting market demand.

The rheological properties of the polysaccharide solution are very important for the food to be applied, and will directly affect the sensory quality of the food. Therefore, when developing a food and choosing a colloid, the rheological properties of the colloid are the first factors to be considered . Yeast mannan with different purity has a large difference in viscosity and has different effects on the application system. How to achieve the functional properties of yeast mannan without changing the rheological properties of the system is very important.

Contents of the invention

In order to solve the problems in the prior art, the object of the present invention is to provide a method for preparing high-purity yeast mannan.

In order to realize the object of the invention, the technical scheme of the present invention is as follows:

A low-viscosity, high-purity yeast mannan preparation method, using the cell wall of Saccharomyces cerevisiae as raw material, adopts water extraction and alcohol precipitation method to obtain crude yeast mannan, which is first enzymatically hydrolyzed by neutral protease, and protected in phosphate buffer , carry out pronase enzymatic hydrolysis and several times of centrifugation, and then process it.

The low-viscosity, high-purity yeast mannan product obtained by the technical scheme of the present invention is a white soft cotton wool-like solid.

Further, the preparation method specifically includes the following steps:

(1) Using the cell wall of Saccharomyces cerevisiae as raw material, the crude yeast mannan I (purity below 80%) is obtained by water extraction and alcohol precipitation;

(2) Using deionized water, the crude yeast mannan obtained in step (1) is prepared into a solution;

(3) adding neutral protease neutrase to the solution obtained in step (2) to carry out enzymolysis, and after the enzymolysis is completed, carry out inactivation to obtain a primary enzymolysis solution of crude yeast mannan;

(4) centrifuging the primary enzymolysis solution obtained in step (3);

(5) Take the supernatant obtained in step (4), add ethanol for alcohol precipitation, and centrifuge to collect the precipitate to obtain the crude yeast mannan II;

(6) Take the precipitate obtained in step (5), add phosphate buffer to prepare a solution, and use NaOH to adjust the pH value of the solution to 7.5-8.0;

(7) Take the solution obtained in step (6), add pronase to carry out enzymolysis, and after the enzymolysis is completed, carry out enzyme inactivation to obtain a secondary enzymolysis solution of crude yeast mannan;

(8) centrifuging the secondary enzymolysis solution gained in step (7);

(9) Take the supernatant obtained in step (8), add ethanol to carry out alcohol precipitation, and centrifuge to collect the precipitate;

(10) dissolving the precipitate obtained in step (9) by deionization, stirring evenly until the dispersion is complete, desalting the solution by ultrafiltration, and collecting the resulting retentate;

(11) Spray-dry the retentate obtained in step (10) to obtain low-viscosity, high-purity yeast mannan III.

Wherein, in the step (1), the crude yeast mannan is extracted by hot water extraction.

Wherein, in the step (2), deionized water is used to prepare the crude yeast mannan obtained in the step (1) into a 15% w/v solution.

Wherein, in the step (3), the amount of neutral protease added is 0.05% of the protein content in the crude yeast mannan, the enzymolysis temperature is 45° C., and the enzymolysis time is 2 hours.

Wherein, in the step (4), the centrifugation speed is 4000-5000 rpm, and the centrifugation time is 5-10 min.

Wherein, in the step (5), the amount of ethanol is twice the volume of the obtained supernatant, the centrifugation speed is 4000-5000 rpm, and the centrifugation time is 10-20 min.

Wherein, in the step (6), the concentration of the solution is 15% w/v, the concentration of the phosphate buffer is 0.02M-0.2M, the pH is 7.0; the concentration of NaOH is 0.01M.

Wherein, in the step (7), the amount of pronase added is 0.05% of the protein content in the crude yeast mannan, the enzymolysis temperature is 40° C., and the enzymolysis time is 8 hours.

Wherein, in the step (8), the centrifugation speed is 4000-5000 rpm, and the centrifugation time is 5-10 min.

Wherein, in the step (9), the amount of ethanol used is three times the volume of the obtained supernatant, the centrifugation speed is 4000-5000 rpm, and the centrifugation time is 10-20 min.

Wherein, in the step (10), the conditions adopted for the ultrafiltration are respectively an ultrafiltration membrane with a molecular weight cut-off of 10 kDa, an operating pressure of 20-25 psi, a feed liquid concentration of 4%-6%, and an operating temperature of normal temperature.

Wherein, in the step (11), the retentate is spray-dried.

The beneficial effects of the present invention are:

1. The process is easy to operate. Compared with the previous purification method through gel filtration chromatography column, the purity of yeast mannan can be quickly improved and the protein content can be reduced by enzymatic hydrolysis coupled with centrifugation. The time required is short and the sample can be obtained at one time. large.

2. The method for improving the purity of yeast mannan of the present invention uses phosphate buffer to prepare the solution, which ensures that the original structure of the polysaccharide is not damaged to the greatest extent during the pH adjustment process.

3. The method for improving the purity of yeast mannan of the present invention adopts enzymatic hydrolysis and centrifugal coupling; its protein removal rate is high, and the loss of polysaccharide is small. The original impurities in the solution and the impurities in the two enzymatic hydrolysis solutions can be removed by centrifugation, which improves the purity of the sample to a greater extent, and can reduce the time required for pronase enzymatic hydrolysis. Compared with the traditional Sevage method, etc., it has the advantages Environmental protection, high efficiency, safety and other advantages, suitable for large-scale production requirements.

4. The method for improving the purity of yeast mannan of the present invention uses the combination of secondary enzymatic hydrolysis, multiple centrifugation, ultrafiltration and spray drying, and the equipment used can meet the needs of industrial production.

5. In the process of the present invention, three kinds of purity yeast mannans are involved in total, and the yeast mannans I and II have higher viscosity and are pseudoplastic fluids, while the final obtained yeast mannans have a low viscosity and a purity greater than 90 %, and it is a Newtonian fluid, which can be used in food systems to improve its functional properties without affecting the original rheological properties of the system.

Description of drawings

Fig. 1 is a schematic flow chart of a method for preparing high-purity yeast mannan according to the present invention.

Fig. 2 is a comparison of the rheological properties of yeast mannan solutions with different purity in the examples of the present invention.

detailed description

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Yeast cell walls in the following examples were purchased from Zhejiang Shenyou Biotechnology Co., Ltd.

The data processing of each step in the following examples is accomplished by using DPS software.

Example 1

A preparation method of high-purity yeast mannan, through the following process steps (as shown in Figure 1):

(1) Using the cell wall of Saccharomyces cerevisiae as raw material, the crude yeast mannan I (purity: 76.60%) was prepared by water extraction and alcohol precipitation;

(2) adding deionized water to dissolve the crude yeast mannan obtained in step (1), and stirring until uniformly dispersed, so that the final concentration of the crude yeast mannan solution is 15% (w/v);

(3) enzymatically treat the solution obtained in step (2) with neutral protease neutrase, the consumption of neutral protease neutrase is 0.05% of the protein content in the crude product yeast mannan obtained in step (1), enzymolysis temperature and time Respectively 45 ℃, 2h;

(4) centrifuging the enzymolysis solution obtained in step (3), the centrifugal speed and time are respectively 4000rpm and 10min;

(5) Take the supernatant obtained in step (4), add twice the volume of ethanol for alcohol precipitation, centrifuge at 4000rpm for 20min to collect the precipitate, and obtain the crude yeast mannan II;

(6) Take the precipitate obtained in step (5), add 0.02M, pH7.0 phosphate buffer to prepare a 15% (w/v) solution, and adjust the pH to 7.8 with 0.01M NaOH;

(7) Get the solution gained in step (6), add pronase for enzymolysis treatment, the consumption of pronase is 0.05% of the protein content in the crude product yeast mannan obtained in step (1), and the enzymolysis temperature and time are respectively 40℃, 8h;

(8) Centrifuge the enzymolysis solution obtained in step (7), the centrifugal speed and time are respectively 4000rpm, 20min;

(9) Collect the supernatant after centrifugation in step (8), add three times the volume of ethanol for alcohol precipitation, and collect the precipitate by centrifugation. The speed and time of centrifugation are respectively 4000rpm and 20min;

(10) Dissolve the precipitate collected in step (9) with deionized water, and use ultrafiltration technology to desalt. The operating pressure is 20 psi, the concentration of the feed solution is 4%, and the operating temperature is normal temperature.

(11) Collect the retentate obtained in step (10) and spray dry it to obtain yeast mannan III with high purity.

The yeast mannan obtained in this example is a white soft cotton wool-like solid. After enzymatic hydrolysis and centrifugal coupling treatment, the purity of the yeast mannan obtained can reach 90.20%, and the protein content is 6.33%. Compared with Ⅰ and Ⅱ, the purity of yeast mannan Ⅲ has been significantly improved, the viscosity has been significantly decreased (p<0.05), and it has changed from a pseudoplastic fluid to a Newtonian fluid (see Figure 2).

Example 2

A preparation method of high-purity yeast mannan, through the following process steps (as shown in Figure 1):

(1) Using the cell wall of Saccharomyces cerevisiae as raw material, the crude yeast mannan I (purity: 77.75%) was prepared by water extraction and alcohol precipitation;

(2) adding deionized water to dissolve the crude yeast mannan obtained in step (1), and stirring until uniformly dispersed, so that the final concentration of the crude yeast mannan solution is 15% (w/v);

(3) enzymatically treat the solution obtained in step (2) with neutral protease neutrase, the consumption of neutral protease neutrase is 0.05% of the protein content in the crude product yeast mannan obtained in step (1), enzymolysis temperature and time Respectively 45 ℃, 2h;

(4) Centrifuge the enzymolysis solution obtained in step (3), the centrifugal speed and time are respectively 5000rpm, 5min;

(5) Take the supernatant obtained in step (4), add twice the volume of ethanol for alcohol precipitation, centrifuge at 5000rpm for 10min to collect the precipitate, and obtain the crude yeast mannan II;

(6) Take the precipitate obtained in step (5), add 0.2M, pH7.0 phosphate buffer to prepare a 15% (w/v) solution, and adjust the pH to 8.0 with 0.01M NaOH;

(7) Get the solution gained in step (6), add pronase for enzymolysis treatment, the consumption of pronase is 0.05% of the protein content in the crude product yeast mannan obtained in step (1), and the enzymolysis temperature and time are respectively 40℃, 8h;

(8) Centrifuge the enzymolysis solution obtained in step (7), the centrifugal speed and time are respectively 5000rpm and 10min;

(9) Collect the supernatant after centrifugation in step (8), add three times the volume of ethanol for alcohol precipitation, and collect the precipitate by centrifugation. The speed and time of centrifugation are respectively 5000rpm and 10min;

(10) Dissolve the precipitate collected in step (9) with deionized water, and use ultrafiltration technology to desalt. The operating pressure is 25 psi, the concentration of the feed solution is 6%, and the operating temperature is normal temperature.

(11) Collect the retentate obtained in step (10) and spray dry it to obtain yeast mannan III with high purity.

The yeast mannan obtained in this example is a white soft cotton wool-like solid. After enzymatic hydrolysis and centrifugal coupling treatment, the purity of the obtained yeast mannan can reach 93.65%, and the protein content is 5.07%. See Table 1. Compared with yeast mannan Ⅰ and Ⅱ, the purity of yeast mannan Ⅲ was significantly improved, the viscosity was significantly decreased (p<0.05), and it changed from pseudoplastic fluid to Newtonian fluid.

Table 1 The main components of yeast mannan

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (7)

1. a kind of preparation method of low viscosity, high-purity yeast mannan, it is characterized in that, described preparation method specifically comprises the steps: (1) using the cell wall of Saccharomyces cerevisiae as raw material, adopting water extraction and alcohol precipitation method to obtain crude yeast mannan; (2) Using deionized water, the crude yeast mannan obtained in step (1) is prepared into a solution; (3) adding neutral protease to the solution obtained in step (2) to carry out enzymolysis, after the enzymolysis is completed, to inactivate the enzyme to obtain a primary enzymolysis solution of crude yeast mannan; (4) centrifuging the primary enzymolysis solution obtained in step (3); (5) Take the supernatant obtained in step (4), add ethanol to carry out alcohol precipitation, and centrifuge to collect the precipitate; (6) Take the precipitate obtained in step (5), add phosphate buffer to prepare a solution, and use NaOH to adjust the pH value of the solution to 7.5-8.0; (7) Take the solution obtained in step (6), add pronase to carry out enzymolysis, and after the enzymolysis is completed, carry out enzyme inactivation to obtain a secondary enzymolysis solution of crude yeast mannan; (8) centrifuging the secondary enzymolysis solution gained in step (7); (9) Take the supernatant obtained in step (8), add ethanol to carry out alcohol precipitation, and centrifuge to collect the precipitate; (10) dissolving the precipitate obtained in step (9) by deionization, stirring evenly until the dispersion is complete, desalting the solution by ultrafiltration, and collecting the resulting retentate; (11) Spray-dry the retentate obtained in step (10) to obtain low-viscosity, high-purity yeast mannan; Wherein, in the step (3), the amount of neutral protease added is 0.05% of the protein content in the crude yeast mannan, the enzymolysis temperature is 45°C, and the enzymolysis time is 2h; In the step (6), the concentration of the solution is 15% w/v, the concentration of the phosphate buffer is 0.02M-0.2M, and the pH is 7.0; the concentration of NaOH is 0.01M; In the step (7), the amount of pronase added is 0.05% of the protein content in the crude yeast mannan, the enzymolysis temperature is 40° C., and the enzymolysis time is 8 hours. 2. The preparation method according to claim 1, characterized in that, in the step (2), deionized water is used to prepare the crude yeast mannan obtained in the step (1) into a 15% w/v solution. 3. The preparation method according to claim 1, characterized in that, in the step (4), the centrifugal speed is 4000-5000 rpm, and the centrifugation time is 5-10 min. 4. The preparation method according to claim 1, characterized in that, in the step (5), the amount of ethanol is twice the volume of the obtained supernatant, the centrifugation speed is 4000-5000 rpm, and the centrifugation time is 10-20 min. 5. The preparation method according to claim 1, characterized in that, in the step (8), the centrifugal speed is 4000-5000 rpm, and the centrifugation time is 5-10 min. 6. according to the described preparation method of claim 1 or 5, it is characterized in that, in described step (9), the consumption of ethanol is the triple volume of gained supernatant, and centrifugation speed is 4000～5000rpm, and centrifugation time is 10～ 20min. 7. the preparation method according to claim 6 is characterized in that, in described step (10), the condition that ultrafiltration adopts is the ultrafiltration membrane that molecular weight cut-off is 10kDa, operating pressure is 20～25psi, feed liquid concentration 4% to 6%, and the operating temperature is normal temperature.